Project Summary Most essential proteins perform their function as part of a protein complex. Many such complexes are large, multi-subunit entities whose structures are well beyond the capabilities of traditional methods of structural determination. This TR&D builds upon previous pioneering achievements by the YRC in developing novel techniques to determine the structure of proteins and protein complexes. We will develop an arsenal of new techniques that will complement current methods of structural determination, empowering the scientific community to address structural questions that were previously out of reach. In Specific Aim 1, we will develop our already highly successful protein cross-linking/mass spectrometry (XL-MS) technology to: (1) Facilitate its adoption throughout the scientific community through development of a quality control toolkit; (2) Increase its sensitivity through improved fragmentation of cross-linked peptides; (3) Incorporate quantitative capabilities allowing XL-MS to be used to study dynamic populations of protein complex conformations and; (4) Develop a comprehensive set of computational tools to identify cross-linked peptides and statistically validate those identifications. In Specific Aim 2, we will develop a complementary method, molecular painting, which will allow the determination of surface-surface interactions in protein complexes ? even in vivo where current techniques such as HD exchange cannot be applied. In our third and final specific aim we will use co-evolution data to model protein structures and interfaces based on covariation of pairs of residues. We will develop technology to apply deep mutational scanning data (a technology developed in the YRC) to model protein interfaces if sufficient sequences are not available for co-evolution methods to be used. We will integrate these predictions with data generated by cross-linking and molecular painting. Through these aims we will drive the field of higher order protein structure determination into the future.